US6291640B1 - Peptidomimetic inhibitors of the human cytomegalovirus protease - Google Patents

Peptidomimetic inhibitors of the human cytomegalovirus protease Download PDF

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US6291640B1
US6291640B1 US09/171,554 US17155498A US6291640B1 US 6291640 B1 US6291640 B1 US 6291640B1 US 17155498 A US17155498 A US 17155498A US 6291640 B1 US6291640 B1 US 6291640B1
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methyl
dimethyl
carboxamido
propyl
phenyl
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Murray D. Bailey
Gulrez Fazal
Pierre Lavallee
William Ogilvie
Marc-Andre Poupart
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Boehringer Ingelheim Canada Ltd
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/10—Tetrapeptides
    • C07K5/1027—Tetrapeptides containing heteroatoms different from O, S, or N
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/20—Antivirals for DNA viruses
    • A61P31/22—Antivirals for DNA viruses for herpes viruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/02—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing at least one abnormal peptide link
    • C07K5/0202—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing at least one abnormal peptide link containing the structure -NH-X-X-C(=0)-, X being an optionally substituted carbon atom or a heteroatom, e.g. beta-amino acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/06—Dipeptides
    • C07K5/06191—Dipeptides containing heteroatoms different from O, S, or N
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/08—Tripeptides
    • C07K5/0827—Tripeptides containing heteroatoms different from O, S, or N
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention relates to compounds, composition and methods for the treatment of human cytomegalovirus (HCMV) infection.
  • HCMV human cytomegalovirus
  • the present invention provides novel peptidomimetic inhibitors of the HCMV protease.
  • HCMV Human Cytomegalovirus
  • the Human Cytomegalovirus is a highly prevalent member of the herpesvirus family infecting up to 80% of the general population. This virus is responsible for opportunistic infections in immunocompromised individuals including organ transplant recipients, cancer patients and AIDS sufferers. Clinical manifestations include disseminated disease, pneumonitis, retinitis and gastro-intestinal infections such as oesophagitis and colitis. Of particular significance are HCMV infections of neonates. This disease is the most common congenitally acquired viral infection in the world. It is estimated that 1% of newborn infants are infected and up to 10% of these are symptomatic and may experience severe complications. Mortality in this latter group approaches 30%.
  • All members of the herpesvirus family express a protein late in the virus life cycle which appears to function as a self assembling scaffold during the manufacture of the viral capsid.
  • This assembly protein is present in immature B-capsids and must be processed to remove a short segment of the C-terminus in order to permit the entry of viral DNA and produce an infectious virus particle. Recently it has been shown that this processing is mediated by a protease which is encoded by the virus.
  • the protease itself is expressed as a precursor protein which is autocatalytically cleaved at least twice (Scheme 1).
  • HCMV protease N o shows significant sequence homology with other herpesvirus proteases. Affinity labeling experiments and site-directed mutagenesis indicate that this enzyme is a serine protease. Recent crystallographic results have shown that HCMV protease represents a novel structure of serine proteases and in fact possesses a unique catalytic triad.
  • HCMV protease While it has not been demonstrated that HCMV protease is absolutely required for viral replication, it has been shown that HSV-1 mutants lacking the analogous enzyme or expressing defective variations of it are unable to grow.
  • the high degree of homology between the proteases of HSV and HCMV support the idea that specific inhibitors of HCMV protease would show antiviral activity and thus have therapeutic value.
  • EP 0,410,411 A2 discloses novel peptidase inhibitors. These peptide analogs all contain a pentafluoroethylketone at P1′, however none of the peptide disclosed contain the amino acid derivatives at P2 disclosed in the present invention.
  • X is CF 3 ; C 2 F 5 ; benzothiazole; oxazolo[4,5b]pyridine; or benzoxazole-R 7 wherein R 7 is H or methyl;
  • X is CF 2 CONH—R 7 , C(O)NH—R 6 ,
  • R 6 is C 0-10 alkyl optionally substituted with phenyl or cyclohexyl, said phenyl or cyclohexyl ring being optionally substituted with Me, halogen —CF 3 , —CH(Me)—C(O)—OBn; —C(O)NH 2 ; or —C(O)-morpholino; said phenyl or cyclohexyl ring optionally fused with a phenyl ring;
  • R 1 is H, Me, or Et
  • R 2 is CH 2 —SO 2 NH 2 ; —C 1-6 alkyl; —(C 1-6 alkyl) aryl; —(C 1-6 alkyl)thiazolo; —CH 2 C(O)—(C 1-6 alkyl); —CH 2 C(O)-pyrrolidino; —CH 2 C(O)-morpholino; —(C 1-6 alkyl)amino; —(C 1-6 alkyl)amido optionally mono- or di-substituted with C 1-6 alkyl, said alkyl optionally substituted with pyridino;
  • W is NH, CH 2 or CH(CH 3 );
  • R 3 is —C 1-12 alkyl; —(C 1-6 alkyl)C(O)OH; or adamantyl;
  • n is 0 or 1
  • R 4 when n is 1, is -C 1-6 alkyl or —(C 1-6 alkyl)-aryl wherein said aryl is optionally substituted with OH;
  • n 0 or 1
  • R 5 when m is 1, is H or —CH 2 OH;
  • Y is H; (CH 2 ) 2 -t-Bu; or an acyl of formula:
  • composition comprising an anti-cytomegalovirus virally effective amount of a compound of formula I or a therapeutically acceptable salt thereof, in admixture with a pharmaceutically acceptable carrier medium or auxiliary agent.
  • An important aspect of the invention involves a method of treating a cytomegalovirus viral infection in a mammal by administering to the mammal an anti-CMV virally effective amount of the compound of formula I or a therapeutically acceptable salt thereof, or a composition as described above.
  • Another important aspect involves a method of inhibiting the replication of cytomegalovirus virus by exposing the virus to a CMV protease inhibiting amount of the compound of formula I or a therapeutically acceptable salt thereof, or a composition as described above.
  • Preferred compounds of the invention include compounds of formula I:
  • z is C. or P.
  • z is C.
  • X is CF 3 ;
  • 2-benzoxazole-R 7 wherein R 7 is H, 4-Me, 5-Me, 6-Me, or 7-Me;
  • R 6 is C 1-7 alkyl, optionally substituted with cyclohexyl, naphtyl, or phenyl optionally substituted with Me, iodo, CF 3 , —CH(Me) —C(O) —OBn; —C(O)NH 2 , or —C(O)-morpholino;
  • X is CF 3 ;
  • R 7 is H, 4-Me, 5-Me, 6-Me, or 7-Me;
  • R 6 is —CH(Me) (CH 2 ) 4 CH 3 ; cyclohexyl; naphtyl; —CH 2 -phenyl; —CH (CH 3 )-phenyl; or —CH(CH 2 CH 3 )-phenyl; —CH 2 -4-iodophenyl; -phenyl-CH 3 ; -phenyl-CF 3 ; -phenyl-C(O)NH 2 ; -phenyl-C(O)-morpholino; -phenyl-CH(Me)—C(O)—OBn; —(CH 2 ) 2 —O—CH 2 -phenyl; —CH 2 -2-benzimidazole; —CH 2 -(3,4-methylenedioxybenzene); or —(CH 2 ) 2 —O—C(O)—OCH 2 CH ⁇ CH 2 ;
  • X is C 2 F 5 ;
  • R 6 is —CH 2 -phenyl; —CH 2 -4-iodophenyl; —CH(CH 3 )-phenyl; or —CH(CH 2 CH 3 ) -phenyl; —CH (Me)-naphtyl; —CH 2 CH(Me)-phenyl; —(CH 2 ) 2 —O—CH 2 -phenyl; —CH 2 -2-benzimidazole; or —CH 2 -(3,4-methylenedioxybenzene);
  • R 1 is H, methyl or ethyl.
  • R 1 is H or methyl.
  • R 1 is H or methyl
  • R 2 is —CH 2 -phenyl
  • R 2 is —CH 2 —C(O)—(N-pyrrolidino);
  • R 2 is —CH 2 —C(O)—(N-pyrrolidino);
  • W is NH or CH 2 .
  • W is NH
  • R 3 is ethyl; isopropyl; t-Bu; CH 2 -t-Bu; or adamantyl.
  • R 3 is ethyl; isopropyl; or t-Bu.
  • R 3 is isopropyl; or t-Bu.
  • n is 0 or 1.
  • n 0.
  • n 1
  • R 4 when n is 1, is isopropyl; t-Bu; or 4-hydroxybenzyl. More preferably, R 4 , when n is 1, is isopropy; or t-Bu. Most preferably, R 4 , when n is 1, is t-Bu;
  • n is 0 or 1. More preferably, m is 0.
  • R 5 when m is 1, is H.
  • Y is H; —CH 2 —CH 2 -t-Bu; or an acyl of formula:
  • Y is H; or an acyl of formula:
  • Y is an acyl of formula:
  • a preferred compound of the invention is selected from the group consisting of:
  • a further aspect of the present invention is a solid phase process for the synthesis of peptidyl activated ketones comprising the steps of:
  • R is a side chain of a natural or non-natural amino acid
  • X′ is CF 3 , CF 2 CONH—R 30 , C(O)NH—R 30 , or C(O)OR 30 , wherein R 30 is a cyclic C 3-12 alkyl or acyclic C 1-10 alkyl or cyclic C 3-12 alkenyl or acyclic C 2-12 alkenyl, said alkyl or alkenyl optionally substituted with NH 2 , OH, SH, halo, or carboxyl;
  • alkyl or alkenyl optionally containing at least one heteroatom independently selected from the group consisting of: O, S, and N; or R 30 is a C 6 or C 10 aryl or C 7-16 aralkyl optionally substituted with C 1-6 alkyl, NH 2 , OH, SH, halo, carboxyl or carboxy(lower)alkyl; said aryl or aralkyl optionally containing at least one heteroatom independently selected from the group consisting of: O, S, and N;
  • A is a divalent spacer group which comprises a non-reactive divalent hydrocarbyl group having from 2 to 15 carbon atoms;
  • Pg is an amino protecting group
  • the cleavage step as herein described is carried out in THF, aq.HCl, and AcOH at a temperature of about 60° C. for about 4 hours; and said resin is filtered at least once.
  • the resin is selected from the group consisting of: polystyrene or pegylated polystyrene functionalized with benzydrylamine (BHA); 4-methyl benzydrylamine (MBHA); and aminomethyl (AM).
  • BHA benzydrylamine
  • MBHA 4-methyl benzydrylamine
  • AM aminomethyl
  • the in situ activation is carried out with the addition of a coupling agent selected from the group consisting of: 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate (TBTU); 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU); diisopropyl carbodiimide (DIC), and dicyclohexyl carbodiimide (DCC).
  • a coupling agent selected from the group consisting of: 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate (TBTU); 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU); diisopropyl carbodiimide (DIC), and dicyclohexy
  • the amino protecting group is selected from the group consisting of: t-butyloxycarbonyl (Boc); 9-fluorenylmethyloxy carbonyl (Fmoc); and allyloxy carbonyl (Alloc).
  • X′ is C(O)NH 2 CH 2 -phenyl or C(O)OCH 2 CH ⁇ CH 2 .
  • R is selected from the group consisting of:
  • A is cyclohexyl, phenyl or benzyl.
  • a further aspect of the present invention is a resin of formula 103 as defined above.
  • a further aspect of the present invention is the use of a resin of formula 103 for the solid phase synthesis of peptidyl activated ketones.
  • FIG. 1 is a Dixon plot for competitive inhibition of compound 76 against HCMV protease.
  • FIG. 2 is a Progress curve for the inhibition of HCMV protease by compound 65.
  • FIG. 3 is a Progress curve for the inhibition of HCMV protease by compound 76.
  • the natural amino acids with exception of glycine, contain a chiral carbon atom. Unless otherwise specifically indicated, the compounds containing natural amino acids with the L-configuration are preferred. However, applicants contemplate that when specified, some amino acids of the formula I can be of either D- or L-configuration or can be mixtures of D- and L-isomers, including 1:1 epimeric mixtures.
  • the non-natural amino acids include, but are not limited to, ⁇ -aminoadipic acid, ⁇ - ⁇ -diamino butyric acid, ornithine, pipecolic acid, sarcosine, thyroxine, hydroxylysine, and hydroxyproline.
  • tert-butylglycine refers to a compound of formula:
  • side chain with reference to an amino acid or amino acid derivative means a residue attached to the ⁇ -carbon atom of the ⁇ -amino acid.
  • the R-group side chain for glycine is hydrogen, for alanine it is methyl, for asparagine it is CH 2 —C(O)NH 2 , for glutamine it is CH 2 CH 2 C(O)NH 2 , and tert-butylglycine it is tert-butyl.
  • R-group side chain for glycine is hydrogen, for alanine it is methyl, for asparagine it is CH 2 —C(O)NH 2 , for glutamine it is CH 2 CH 2 C(O)NH 2 , and tert-butylglycine it is tert-butyl.
  • halo as used herein means a halogen radical selected from bromo, chloro, fluoro or iodo.
  • C 1-10 alkyl or “(lower)alkyl” as used herein, either alone or in combination with another radical, means cyclic or acyclic (meaning straight chain or branched) alkyl radicals containing up to ten carbon atoms and includes, for example, methyl, ethyl, propyl, butyl, hexyl, 1-methylethyl, 1-methylpropyl, 2-methylpropyl, 1,1-dimethylethyl.
  • the alkyl radical will contain at least 3 carbon atoms.
  • C2-10 alkenyl as used herein, either alone or in combination with another radical, means an alkyl radical as defined above containing from 2 to 10 carbon atoms, and further containing at least one double bond.
  • alkenyl includes allyl.
  • C 6 or C 10 aryl as used herein, either alone or in combination with another radical, means either an aromatic monocyclic system containing 6 carbon atoms or an aromatic bicyclic system containing 10 carbon atoms.
  • aryl includes phenyl or naphthalene.
  • C 7-16 aralkyl as used herein, either alone or in combination with another radical, means an aryl as defined above linked through an alkyl group, wherein alkyl is as defined above containing from 1 to 6 carbon atoms.
  • Aralkyl includes for example benzyl, and butylphenyl.
  • divalent spacer group means a non-reactive divalent hydrocarbyl group from 2 to 15 carbon atoms and includes, but is not limited to, cyclohexane, phenyl and benzyl.
  • heterocycle as used herein, either alone or in combination with another radical, means a monovalent radical derived by removal of a hydrogen from a five-, six-, or seven-membered saturated or unsaturated heterocycle containing from one to four heteroatoms selected from nitrogen, oxygen and sulfur.
  • suitable heterocycles include pyrrolidine, pyridine, thiazole, thiazolidine, benzothiazole, benzoxazole, benzimidazole, and 3,4-methylenedioxybenzene.
  • HCMV protease inhibitors The antiviral activity of the aforementioned peptidomimetic inhibitors of HCMV protease (HCMV protease inhibitors) can be demonstrated by biochemical, microbiological and biological procedures. For example, an assay based on the evaluation of the ability of the test compound to inhibit HCMV protease, an enzyme vital for viral replication.
  • the HCMV protease inhibitor When employed as an antiviral agent, it is administered orally, or systemically to humans in a vehicle comprising one or more pharmaceutically acceptable carriers, the proportion of which is determined by the solubility and chemical nature of the compound, chosen route of administration and standard biological practice.
  • a vehicle comprising one or more pharmaceutically acceptable carriers, the proportion of which is determined by the solubility and chemical nature of the compound, chosen route of administration and standard biological practice.
  • the compound or a therapeutically acceptable salt thereof can be formulated in unit dosage forms such as capsules or tablets each containing a predetermined amount of the active ingredient, ranging from about 50 to 500 mg, in a pharmaceutically acceptable carrier.
  • the HCMV protease inhibitor is administered by either intravenous, subcutaneous or intramuscular injection, in compositions with pharmaceutically acceptable vehicles or carriers.
  • pharmaceutically acceptable vehicles or carriers for parenteral administration, it is preferred to use the compounds in solution in a sterile aqueous vehicle which may also contain other solutes such as buffers or preservatives as well as sufficient quantities of pharmaceutically acceptable salts or of glucose to make the solution isotonic.
  • Suitable vehicles or carriers for the above noted formulations are described in standard pharmaceutical texts, e.g. in “Remington's The Science and Practice of Pharmacy”, 19th ed., Mack Publishing Company, Easton, Pa., 1995, or in “Pharmaceutical Dosage Forms And Drugs Delivery Systems”, 6th ed., H. C. Ansel et al., Eds., Williams & Wilkins, Baltimore, Md., 1995.
  • the dosage of the HCMV protease inhibitor will vary with the form of administration and the particular active agent chosen. Furthermore, it will vary with the particular host under treatment. Generally, treatment is initiated with small increments until the optimum effect under the circumstance is reached. In general, the inhibitor compound is most desirably administered at a concentration level that will generally afford antivirally effective results without causing any harmful or deleterious side effects.
  • the HCMV protease inhibitor is administered in the range of 20 to 200 mg per kilogram of body weight per day, with a preferred range of 25 to 100 mg per kilogram.
  • the HCMV protease inhibitor is administered either topically or intraocularly (injection or implant) in a suitable preparation.
  • a suitable preparation for example, an implant containing the compound in a suitable formulation can be surgically placed in the posterior segment of the eye through a small incision.
  • the HCMV protease inhibitor is administered at a dosage of 10 mg to 150 mg per kilogram of body weight per day, although the aforementioned variations will occur. However, a dosage level that is in the range of from about 10 mg to 100 mg per kilogram of body weight per day is most desirably employed in order to achieve effective results.
  • Inhibitors containing a trifluoromethyl ketone function were obtained in one of three ways: solution chemistry or solid phase synthesis: schemes 2 or 3.
  • Inhibitors containing an ⁇ -ketoamide were obtained in one of two ways: solid phase: scheme 3 or solution chemistry: scheme 4.
  • Inhibitors containing other activated ketones were obtained by solution chemistry: scheme 5.
  • Inhibitors which incorporate an asparagine residue at P 2 could be prepared through solid phase synthesis using the asparagine side chain as an attachment point to the resin (Abraham, N. A.; Fazal, G.; Ferland, J.-M.; Rakhit, S.; Gauthier, J., A new solid phase strategy for the synthesis of mammalian glucagon, Tetrahedron Lett . 1991, 32, 577-580).
  • peptidyl trifluoromethyl ketone or ⁇ -ketoamide is typically performed in solution, by preparing a precursor alcohol and submitting it to a final oxidation step.
  • This oxidation is often problematic (especially when other oxidizable groups are present in the molecule) and sometimes limits the choice of pharmacophore to be incorporated in the inhibitor.
  • the protected semicarbazide 110 was deprotected and neutralized.
  • the resulting semicarbazide was then condensed in refluxing toluene, with the activated ketone 109 under acid catalysis and azeotropic removal of water, to give the trans semicarbazone 112 in moderate yield (in the case of the ketoamide, a cis/trans mixture was obtained).
  • the hydrogenolysis of the benzyl ester proceeded without problem to give the corresponding acid 113 in quantitative yield.
  • the acid was then coupled to a polystyrene BHA resin by in situ activation with TBTU followed by the removal of the Boc protecting group to give the desired resin 103.
  • the final cleavage from the polymer support was performed by refluxing the dried resin in a THF solution containing aqueous HCl and acetic acid at 65° C. In order to maximize the yields, we found it necessary to filter the resin and repeat this protocol once more. In general, the cleavage from the resin was slightly slower for the valine-derived trifluoromethyl ketones 212-217 (see Example 62) than for their alanine (201-207) or ethyl glycine (208-211) counterpart. This difference in rate of hydrolysis could be compensated for by doing one extra cleavage for valine derivatives.
  • nucleophilic or oxidizable side chains such as the ones present in serine, methionine, tyrosine, histidine, lysine or aspartic acid.
  • the cleavage of inhibitors containing an asparagine residues adjacent to the trifluoromethyl group was more problematic. In this case, it was found necessary to use the N-trityl protected asparagine and to deprotect the trityl group in solution after the cleavage from the resin. The presence of an asparagine residue elsewhere in the sequence did not however necessitate any side chain protection.
  • the cleavage conditions were mild enough to be compatible with various acid-sensitive protecting groups such as N-Boc, O-t-Bu ether, O-t-Bu ester and O-Bn ester. During the cleavage, methyl esters are however hydrolyzed to an extent of 50%. In most cases, the treatment with 75% TFA prior to the cleavage from the resin, was sufficient to completely deprotect the acid-sensitive side chain protecting groups. However, in a few examples the O-t-Bu derivative of threonine and aspartic acid were also isolated indicating that the deprotection was not complete in those cases.
  • ⁇ -ketoamides could also be synthesized by the same process (compounds 218 and 219 from example 62) and in similar overall yields.
  • Peptides containing activated ketones other than trifluoromethyl ketone could also be prepared by sequentially coupling a suitably protected amino alcohol with the required amino acids or peptide segment using standard solution methods. After the complete backbone was established, oxidation of the resulting alcohol gave the desired compound.
  • the preparation of the various building blocks are shown in Schemes 4, 5 and 6.
  • the ⁇ , ⁇ -difluoroamide 13 was prepared from an ultrasonic Reformatsky reaction (Thaisrivongs, S.; Pals, P. T.; Kati, W. M.; Turner, S. R.; Thomasco, L. M.; Watt, W.; Design and synthesis of potent and specific renin inhibitors containing diflurostatine, difluorstatone, and related analogues. J. Med. Chem . 1986, 24, 2080-2087) between ethyl bromodifluoroacetate and Boc-alaninal followed by treatment with benzylamine.
  • Benzothiazole 14 was obtained in a straightforward manner when 2-lithiobenzothiazole was added to this same aldehyde.
  • 23 could be hydrogenated to the corresponding acid and the appropriate P1′ amine coupled. Coupling the required amino acids using standard methods gave 25. The alcohol 25 was readily oxidized to the desired ketones as above.
  • oxazolidinone 29 was obtained from acid 27 using procedures described previously (Gage, J. R.; Evans, D. A. Diastereoselective aldol condensation using a chiral oxazolidinone auxiliary: (2S*, 3S*)-3-hydroxy-3-phenyl-2-methylpropanoic acid. Org Syn . 1989, 68, 83-91). Formation of the enolate followed by treatment with TrisN 3 (Evans, D. A.; Britton, T. C.; Ellman, J. A.; Dorow, R. L. The asymmetric synthesis of ⁇ -amino acids.
  • Analytical HPLC were carried out on the following systems; System A: Vydac C18, 10 ⁇ m analytical column (24 cm ⁇ 4.6 mm); mobile phase, acetonitrile/0.06% trifluoroacetic acid (TFA) in water/0.06% TFA; System B: Vydac C18, 5 ⁇ m analytical column (15 cm ⁇ 4.6 mm); mobile phase, acetonitrile in 50 mM NaH 2 PO 4 at pH 4.4; System C: Vydac C 8, 10 ⁇ m analytical column (24 cm ⁇ 4.6 mm); mobile phase, acetonitrile in 20 mM Na 2 HPO 4 at pH 8.0; System D: symmetry shield C8, 10 ⁇ m analytical column (15 cm ⁇ 3.9 mm); mobile phase, acetonitrile in 20 mM Na 2 HPO 4 at pH 9.0; System E: Supelcosil C8, 5 ⁇ m analytical column (15 cm ⁇ 4.6 mm); mobile phase, acetonitrile/0.1% TFA in water/0.1% TFA
  • hydrochloride salt Fmoc, 9-fluorenylmethyloxycarbonyl; HCMV, human cytomegalovirus; HOBt, 1-hydroxybenzotriazole hydrate; MES, 4-morpholineethanesulfonic acid; NMP, N-methylpyrrolidone; PCR, polymerase chain reaction; Ph, phenyl; PMSF, phenylmethylsulfonyl fluoride; QSAR, quantitative structure activity relationship; Tbg, tertiary- butylglycine; tBu, tertiary-butyl; TFA, trifluoroacetic acid; Trt, triphenylmethyl; TBTU, O-(benzotriazol-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate; TCEP, tris(2-carboxyethyl)phosphine hydrochloride; TRIS, tris(hydroxymethyl)aminomethane.
  • the peptides were assembled on a ACT396 peptide synthesizer sold by Advanced Chemtech (Louisville, Ky.). Each reaction vessels were charged with the appropriate resins 103 (0.25 mmol) and were successively washed with 3.5 mL portions of CH 2 Cl 2 (2 ⁇ ), MeOH (2 ⁇ ) and CH 2 Cl 2 (2 ⁇ ).
  • the amino acids were coupled as their activated HOBt esters, utilizing 4.8 equivalents of the reagents as follows: A 0.5 M solution of a mixture of Fmoc-protected amino acid and HOBt in DMF (2.4 mL, 1.2 mmol of each) was added to the deprotected resin, followed by addition of a 0.5 M DIC solution in CH 2 Cl 2 (2.4 mL, 1.2 mmol). The reaction vessel was shaken for 3.5 h. The reaction vessel was drained and the remaining resin was washed twice with 5 mL of CH 2 Cl 2 . Fresh portions of reagent solutions were added and the coupling step was repeated for 3.5 h.
  • the peptides were assembled as above except for the coupling which was done as follow:
  • the resin was suspended in NMP (0.35 mL) and was treated with a 0.5 M solution of a mixture of Fmoc-protected amino acid and HOBt in NMP (1.8 mL, 0.9 mmol of each), a 0.5 M solution of TBTU in DMF (1.8 mL, 0.9 mmol) and a 1.0 M solution of DIPEA in NMP (1.8 mL. 1.8 mmol).
  • the reaction vessel was shaken for 1.25 h, it was drained and the remaining resin was washed twice with 3.5 mL of DMF. Fresh portions of reagent solutions were added and the coupling step was repeated for 1.25 h.
  • the deprotection of the Fmoc group was done by treating the resin with a 25% solution of piperidine in DMF for 25 minutes.
  • the peptides were assembled on a COUPLERTM 250 C (VEGA Biotechnologies) or on an ACT 90 (Advanced ChemTech) peptide synthesizer.
  • the reaction vessel was charged with the appropriate resins 103 (0.25 mmol) which was successively washed with 15 mL portions of CH 2 Cl 2 (2 ⁇ , MeOH (2 ⁇ ) and CH 2 Cl 2 (2 ⁇ ).
  • the amino acids were coupled as their activated HOBt esters, utilizing 3 equivalents of the reagents as follows: The resin was suspended in DMF (15 mL) and was treated with the Boc-protected amino acid (0.75 mmol), HOBt hydrate (0.75 mmol), DIPEA (1.5 mmol, 0.26 mL) and TBTU (0.75 mmol, 241 mg). The reaction vessel was shaken for 1 h and the completion of the coupling monitored by Kaiser test. In the case of incomplete couplings, the reaction vessel was drained and the resin was washed twice with 15 mL of CH 2 CL 2 . Fresh reagents were added and the coupling step was repeated for an extra hour.
  • the reaction vessel was drained and the resin washed as above.
  • the Boc protecting group was removed by successive treatment (5 min. then 20 min.) with 15 mL of a 45% solution of TFA in CH 2 Cl 2 .
  • the resin was washed with CH 2 CL 2 (2 ⁇ ), 5% DIPEA in CH 2 CL 2 (1 min. then 5 min.), CH 2 Cl 2 (2 ⁇ ), MeOH (2 ⁇ ) and CH 2 Cl 2 (2 ⁇ ).
  • the dried resin ( ⁇ 800 mg) was suspended in THF (9 mL), H 2 O(0.50 mL), AcOH (0.25 mL) and 1M aq. HCl (0.10 mL) and was heated in a bomb at 65° C. for four hours. The solution was cooled down, filtered and treated as above one more time. In the case where the sequence contained a basic residue such as lysine or histidine, an extra 0.05 mL of 1 M HCl was used and the procedure was repeated a third time.
  • the ⁇ -hydroxy benzyl ester peptides were purified by flash chromatography.
  • the acid 24 was then obtained from the benzyl ester (1.10 g, 2.0 mmol) by hydrogenation over 10% Pd/C (55 mg) in ethanol (30 mL) at atmospheric pressure over the course of a few hours to afford after filtration through a pad of Celite a white solid (0.95 g, 100% yield).
  • This compound was prepared according to a literature procedure analogous to the preparation of the valine analogue (Skiles, J. W.; Fuchs, V.; Miao, C.; Sorcek, R.; Grozinger, K. G.; Mauldin, S. C.; Vitous, J.; Mui, P. W.; Jacober, S.; Chow, G.; Matteo, M.; Skoog, M.; Weldon, S. T.; Possanza, G.; Keirns, J.; Letts, G.; Rosenthal, A. Inhibition of human leukocyte elastase (HLE) by N-substituted peptidyl trifluoromethyl ketones. J.
  • the reaction was stirred at ⁇ 78° C. for 90 min and then allowed to warm to ⁇ 30° C. for 2 h.
  • the reaction was quenched by the addition of sat. NH 4 Cl (125 mL) .
  • the organic phase was washed with H 2 O (2 ⁇ 50 mL), dried over Na 2 SO 4 , filtered and concentrated to provide an orange oil.
  • the oil was redissolved in 20% MeOH/THF (100 mL) and transferred to a 500 mL round bottom flask. The solution was cooled to 0° C. before NaBH 4 (1.9g, 50.1 mmole) was added portionwise over 5 min (Caution! foaming occurs).
  • the reaction was subsequently stirred for 1 h at 0° C.
  • This compound was prepared from methyl ester 12 using the procedure previously described (Tsutsumi, S.; Okonogi, T.; Shibahara, A.; Ohuchi, S.; Hatsushiba, E.; Patchett, A. A.; Christensen, B. G. Synthesis and 20 structure-activity relationships of peptidyl ⁇ -keto heterocycles as novel inhibitors of prolyl endopeptidase. J. Med. Chem . 1994, 37, 3492-3502).
  • This compound was prepared from 15 and 2-aminophenol using the procedure previously described ((a) Edwards, P. D.; Meyer, E. F. Jr.; Vijayalakshmi, I.; Tuthill, P. A.; Andisik, D. A.; Gomes, B.; Strimpler, A. Design, synthesis, and kinetic evaluation of a unique class of elastase inhibitors, the peptidyl ⁇ -ketobenzoxazoles, and the X-ray crystal structure of the covalent complex between porcine pancreatic elastase and Ac-Ala-Pro-Val-2-benzoxazole. J. Am. Chem. Soc . 1992, 114, 1854-1863).
  • This material was prepared as a 1:1 mixture of isomers in 12% yield from the above cyanohydrin (978 mg, 4.18 mmol) and 2-amino-3-hydroxypyridine (505 mg, 4.60 mmol) using the procedure described above for compound 16.
  • An analytical sample was obtained by recrystallization from EtOAc in hexanes (one isomer).
  • This material was prepared as a 1:1 mixture of isomers in 35% yield from the above cyanohydrin (707 mg, 3.02 mmol) and 2-amino-m-cresol (409 mg, 3.32 mmol) using the procedure described above for compound 16.
  • An analytical sample was obtained by recrystallization from EtOAc in hexanes (1.3:1 mixture of isomers).
  • This material was prepared as a 1:1 mixture of isomers in 53% yield from the above cyanohydrin (1.10 g, 4.70 mmol) and 2-amino-p-cresol (636 mg, 5.17 mmol) using the procedure described above for compound 16.
  • An analytical sample was obtained by recrystallization from EtOAc in hexanes (7 : 1 mixture of isomers).
  • This material was prepared as a 1:1 mixture of isomers in 71% yield from the above cyanohydrin (1.44 g, 6.15 mmol) and 6-amino-m-cresol (832 mg, 6.77 mmol) using the procedure described above for compound 16.
  • An analytical sample was obtained by recrystallization from EtOAc in hexanes (8:1 mixture of isomers).
  • This material was prepared as a 1:1 mixture of isomers in 57% yield from the above cyanohydrin (609 mg, 2.60 mmol) and 6-amino-o-cresol (330 mg, 2.60 mmol) using the procedure described above for compound 16.
  • An analytical sample (1.2:1 mixture of isomers) was obtained by flash chromatography (30% EtOAc in hexanes).
  • the oxazolidinone 29 (4.2 g, 13.7 mmol) was dissolved in THF (15 mL) and was added dropwise over 15 min to a solution of potassium bis(trimethylsilyl)amide (20.1 mL, 0.69 M in THF, 13.9 mmol) at ⁇ 78° C. After 45 min at ⁇ 78° C., 2,4,6-triisopropylbenzenesulfonyl azide (4.9 g, 15.8 mmol) in THF (10 mL) at ⁇ 78° C. was added in one portion to the enolate.
  • n-Butyllithium (2.4 mL, 1.6 M in hexanes, 3.9 mmol) was added dropwise to a solution of (4S)-( ⁇ )-4-isopropyl-2-oxazolidinone (0.5 g, 3.9 mmol) in THF (5 mL) at ⁇ 40° C. under an atmosphere of nitrogen. After 30 min at ⁇ 40° C., the reaction mixture was cooled to ⁇ 78° C. and 2,2-dimethylsuccinic anhydride (0.5 g, 3.9 mmol) dissolved in THF (2 mL) was added dropwise. The mixture was then stirred magnetically at 0° C. for 1 h.
  • the oxazolidinone 34 (8.67 g, 24.9 mmol) was dissolved in THF (27 mL) and was added dropwise over 15 min to a solution of potassium bis(trimethylsilyl)amide (36.5 mL, 0.69 M in THF, 25.2 mmol) at ⁇ 78° C. After 45 min at ⁇ 78° C., 2,4,6-triisopropylbenzenesulfonyl azide (8.89 g, 28.7 mmol) in THF (15 mL) at ⁇ 78° C. was added in one portion to the enolate.
  • Tetrahydrofuran was evaporated and the aqueous phase was extracted with chloroform (continuous liquid-liquid extraction, 24 h). The aqueous phase was then acidified with concentrated HCl at 0° C., and extracted with EtOAc (3 times). The combined EtOAc extracts were dried (MgSO 4 ), filtered and concentrated.
  • This compound was prepared by the same procedure as for 3 (Example 3), except 1-nitroethane was replaced by 1-nitrobutane. Standard solution coupling conditions were used to prepare the peptide inhibitor with final oxidation of the trifluoromethyl alcohol using Moffatt-Pfitzner method. Purification was performed by preparative HPLC.
  • This compound was prepared in solution using standard coupling methods.
  • the ⁇ , ⁇ -dimethyl aspartic acid residue was incorporated as the ⁇ -benzyl ester derivative.
  • Oxidation of the trifluoromethyl alcohol was accomplished with the Dess-Martin periodinane. Final purification was performed by preparative HPLC.
  • This compound was prepared by solid phase using the activated ketone resin (Example 1).
  • the final reductive amination on the terminal t-butyl glycine amine (0.3 mmol) was performed on solid phase by addition of 3,3-dimethylbutyraldehyde (376 mL, 3.0 mmol) in DMF (15 mL with acetic acid (150 mL), and NaBH 3 CN (63 mg, 1 mmol) for 20 h. After removal of the solvent, the resin was cleaved in the usual fashion. After purification by preparative HPLC the compound was obtained as a white solid (20.6 mg) after lyophilization.
  • This compound was prepared in solution using standard coupling methods.
  • the 2-hydroxy isobutyric acid moiety was introduced as the acetyl derivative.
  • Oxidation of the trifluoromethyl alcohol with the Dess-Martin periodinane was followed by cleavage of the acetate group with aq. NaOH.
  • Final purification was performed by preparative HPLC.
  • the dipeptide (0.43 g, 0.99 mmol) was treated with 4 N HCl/dioxane (10 mL) for 2 h before being concentrated in vacuo.
  • the resulting hydrochloride salt (0.99 mmol) was combined with Boc-Tbg-OH (0.254 g, 1.1 mmol), BOP (0.487 g, 1.1 mmol), and i-Pr 2 NEt (0.52 mL, 3.0 mmol) in CH 2 Cl 2 (10 mL). After 2.5 h at rt, the mixture was extracted into EtOAc and washed with 1 N HCl, saturated aqueous NaHCO 3 , and brine.
  • This peptide (0.30 g, 0.67 mmol) was then treated with 4 N HC l/dioxane ( 10 mL) and concentrated in vacuo.
  • the hydrochloride salt was combined with tert-butylacetic acid (0.094 ⁇ L, 0.74 mmol), BOP (0.33 g, 0.74 mmol), i-Pr 2 NEt (0.23 mL, 1.34 mmol) in CH 2 Cl 2 (10 mL) and stirred 3.5 h at rt.
  • the mixture was diluted with EtOAc and washed with 1 N HCl, saturated aqueous NaHCO 3 , and brine.
  • the organic phase was dried (MgSO 4 ), filtered and concentrated in vacuo.
  • This material (0.27 g, 0.54 mmol) was treated with 4 N HCl/dioxane (6 mL) for 30 min before being concentrated in vacuo.
  • the hydrochloride salt (0.54 mmol) was combined with Boc-Tbg-OH (0.125 g, 0.54 mmol), BOP (0.286 g, 0.65 mmol) and i-Pr 2 NEt (0.23 mL, 1.35 mmol) in CH 2 Cl 2 (3 mL) and stirred for 4 h.
  • the mixture was diluted with EtOAc and washed sequentially with 1 N HCl, saturated aqueous NaHCO 3 , and brine before being dried (MgSO 4 ), filtered and concentrated in vacuo.
  • This peptide (0.25 g, 0.41 mmol) was treated with 4 N HCl/dioxane (3 mL) and stirred 1 h before being concentrated in vacuo.
  • the hydrochloride salt (0.41 mmol) was combined with tert-butylacetic acid (52 ⁇ L, 0.41 mmol), BOP (0.216 g, 0.49 mmol) and i-Pr 2 NEt (0.18 mL, 1.02 mmol) in CH 2 Cl 2 (3 mL) and stirred 4 h.
  • the peptide (0.15 g, 0.245 mmol) was dissolved in CH 2 Cl 2 (15 mL) and treated with Dess-Martin periodinane (0.10 g, 0.245 mmol) and stirred at rt for 5 h.
  • the mixture was diluted with EtOAc and treated with a 1:1 mixture of 10% Na 2 S 2 O 3 : saturated NaHCO 3 (15 min).
  • the organic phase was washed sequentially with saturated NaHCO 3 , 10% citric acid, and brine before being dried (MgSO 4 ), filtered, and concentrated in vacuo.
  • the final product was purified by preparative HPLC to give, after lyophilization, compound 75 as a white solid (0.115 g, 77%).
  • This product was stirred in a mixture of CH 2 Cl 2 (8 mL) and TFA (2 mL) for 2 h. After removal of the solvent, residual TFA was removed by azeotropic distillation with benzene using a rotary evaporator. The residue was dissolved in CH 2 Cl 2 (6 mL) and Boc-Tbg-OH (188 mg, 0.81 mmol), HOBt (209 mg, 1.55 mmol), i-Pr 2 NEt (0.54 mL, 3.10 mmol) and EDC (161 mg, 0.84 mmol) were added. Additional i-Pr 2 NEt was introduced to bring the pH above 8 and stirring was continued overnight.
  • the organic phase was dried (MgSO 4 ), filtered and concentrated in vacuo to give 0.975 g of an amorphous solid.
  • the product was purified by flash chromatography (gradient 15-30% i-PrOH/hexane) to yield the coupled phosphonate derivative as an amorphous solid (0.81 g, 81%).
  • This material (0.75 g, 1.44 mmol) was treated with 4 N HCl/dioxane (30 min) before being concentrated in vacuo.
  • the hydrochloride salt (1.44 mmol) was combined with Boc-Tbg-OH (0.40 g, 1.73 mmol), TBTU (0.555 g, 1.73 mmol) and i-Pr 2 NEt (1.05 mL, 6.05 mmol) in DMF (8 mL) initially at 0° C. (15 min) and then at rt 16 h.
  • the reaction mixture was diluted with EtOAc and washed sequentially with 5% aqueous NaHCO 3 , 1 M citric acid, and brine.
  • This compound (0.70 g, 1.0 mmol) was treated with 4 N HCl/dioxane (30 min) before being concentrated in vacuo.
  • the hydrochloride salt (1.0 mmol) was combined with tert-butylacetic acid (191 ⁇ L, 1.50 mmol), TBTU (0.385 g, 1.20 mmol) and i-Pr 2 NEt (0.52 mL, 3.0 mmol) in DMF (10 mL) for 16 h.
  • the reaction mixture was diluted with EtOAc and washed sequentially with 5% aqueous NaHCO 3 , 1 M citric acid, and brine.
  • the organic phase was dried (MgSO 4 ), filtered and concentrated in vacuo.
  • ketomethylene intermediate (2.42 g, 5.24 mmol, benzyl 6,6-dimethyl-(2S)-2-(2-dimethylamino-2-oxoethyl)-(5S)-5-[(tert-butoxycarbonyl)amino]-4-oxoheptanoate prepared according to Moss et al., (J. Med. Chem., 1996, 39, 4173-4180) in 4N HCl/dioxane (30 mL) was stirred at ambient temperature for 1.5 h.
  • the peptidyl trifluoromethyl ketones and ⁇ -ketoamides of a wide chemical diversity were obtained in 12%-37% overall yield from the corresponding starting resin 103 described in Example 1.
  • the crude material which typically showed an homogeneity of 60-80% by reversed phase HPLC could easily be purified by semi-preparative HPLC. Since the trifluoromethyl ketone and ⁇ -ketoamide fragments 109 were racemic, the desired inhibitors were usually isolated as a 1:1 mixture of diastereomers. In some cases each isomer could be separated during the purification but in most cases, the inhibitors were subjected to biological testing as a mixture of isomers at the activated ketone center.
  • compound 218 was synthesized in the following manner:
  • the IC 50 of compound 218 was found to be 9.4 ⁇ M.
  • a protocol adaptable to a 96-well plate format was designed for the determination of IC 50 values of inhibitors.
  • 125 nM HCMV N o protease was pre-incubated for 5 hr at 30° C. with a range of sequentially diluted inhibitor concentrations (300 to 0.06 ⁇ M depending on the potency of each compound). After this period, enzymatic hydrolysis was initiated by addition of the fluorogenic substrate and the reaction was performed for 2 hr at 30° C. ( ⁇ 30% conversion). No quenching was required before fluorescence measurement since the total scanning time by the plate reader accessory was brief relative to the duration of the reaction.
  • the incubation buffer (essentially similar to the pre-incubation buffer) contained 50 mM Tris/HCl pH 8, 0.5M Na 2 SO 4 , 50 mM NaCl, 0.1 mM EDTA, 1 mM TCEP, 3% v/v DMSO and 0.05% w/v Casein.
  • the final concentrations of HCMV N, protease (expressed in terms of total monomer concentration) and substrate were 100 nM and 5 ⁇ M respectively.
  • IC 50 values were obtained through fitting of the inhibition curve to a competitive inhibition model using SAS NLIN procedure.
  • the mode of inhibition was determined by measurements of the initial rates (in cuvettes) at various substrate (Abz-Tbg-Tbg-Asn(Me) 2 -Ala-SSRLY(3-NO 2 )R-OH) and inhibitor concentrations using the same conditions as above. Data was plotted according to the Cornish-Bowden method ([S]/v versus [I]) and Dixon method (1/v versus [I]) to visually assess the type of inhibition (Cornish-Bowden, A. A simple graphical method for determining the inhibition constants of mixed, uncompetitive and non-competitive inhibitors. Biochem. J . . 1974, 137, 143-144).
  • the specificity of the compounds was determined against a variety of serine proteases (Human leukocyte and porcine pancreatic elastases (HLE & PPE), bovine pancreas ⁇ -chymotrypsin) and one cysteine protease (Human liver cathepsin B).
  • HLE & PPE Human leukocyte and porcine pancreatic elastases
  • bovine pancreas ⁇ -chymotrypsin bovine pancreas ⁇ -chymotrypsin
  • cysteine protease Human liver cathepsin B
  • pNA calorimetric p-nitroanilide
  • Substrate concentrations were kept as low as possible compared to KM to reduce substrate competition. Compound concentrations varied from 300 to 0.06 ⁇ M depending on their potency.
  • the final conditions for each assay were as followed: 50 mM Tris/HCl pH 8, 0.5 M Na 2 SO 4 , 50 mM NaCl, 0.1 mM EDTA, 3% DMSO, 0.01% Tween-20 with [100 ⁇ M Succ-AAPF-pNA and 250 pM ⁇ -chymotrypsin], [133 ⁇ M Succ-AAA-pNA and 8 nM porcine elastase], or [133 ⁇ M Succ-AAV-pNA and 8 nM leukocyte elastase].
  • the compound with its incorporated iodine atom has the added benefit of being a useful compound for X-ray crystallographic studies.
  • Table 8 illustrates further compounds synthesized according to the present invention:
  • Inhibitors 74 and 76 showed increases in potency by factors of ten and five respectively.
  • FIG. 1 shows a Dixon plot obtained for compound 76 which clearly demonstrates that this compound was a competitive inhibitor of HCMV protease.
  • Compound 74 showed slow binding behavior intermediate between that of 76 and 65, while 77 gave a progress curve comparable to 76.
  • the very slow turnover rate shown by HMCV protease, coupled with slow binding kinetics for the present series of inhibitors has implications for the reliability of the enzymatic data.
  • ⁇ -ketobenzoxazole 77 which was actually seven fold more potent against PPE than against HCMV protease.
  • Benzothiazole 80 proved to be a potent inhibitor of HCMV (IC 50 1.1 ⁇ M) and also interacted strongly with PPE (IC 50 9 ⁇ M)
  • Compound 81 was not an inhibitor of PPE but this specificity improvement was accompanied by an 18 fold loss in activity towards HCMV protease.
  • the various methylated benzoxazoles 82-85 were all more potent inhibitors of PPE than of HCMV protease.
  • Compound 76 represented one of the most potent inhibitors of HCMV protease described so far. This structure also suggested the possibility of further increasing potency by extending the C-terminal amide moiety of this inhibitor into the S 1 ′ binding pocket of the enzyme. The observation that the P 1 ′ amino acids are fairly conserved (alanine or serine) prompted us to extend the C-terminus of the ⁇ -ketoamide class of inhibitors in order to try to take advantage of interactions in the S′ pocket.
  • Table 8 compounds 301 to 312 summarize different substitutions of the P2 side-chain that gave potent inhibitors. These include various asparagine amide substitutions and a novel sulfonamide residue.

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US6599940B2 (en) 2000-09-13 2003-07-29 Georgetown University Synthesis of 2-hydroxymethylglutamic acid and congeners thereof
US6649593B1 (en) * 1999-10-13 2003-11-18 Tularik Inc. Modulators of SREBP processing
US20040092429A1 (en) * 2002-01-29 2004-05-13 Zealand Pharma A/S Compositions and methods for modulating connexin hemichannels
US6774212B2 (en) * 1999-12-03 2004-08-10 Bristol-Myers Squibb Pharma Company Alpha-ketoamide inhibitors of hepatitis C virus NS3 protease

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TW200404789A (en) * 1999-03-15 2004-04-01 Axys Pharm Inc Novel compounds and compositions as protease inhibitors
US7244721B2 (en) 2000-07-21 2007-07-17 Schering Corporation Peptides as NS3-serine protease inhibitors of hepatitis C virus
PL206255B1 (pl) 2000-07-21 2010-07-30 Dendreon Corporationdendreon Corporation Inhibitor proteazy wirusa zapalenia wątroby C, zawierająca go kompozycja farmaceutyczna i zastosowanie inhibitora do wytwarzania leku do leczenia chorób związanych z HCV oraz zastosowanie do wytwarzania kompozycji do stosowania w kombinowanej terapii
HUP0303358A3 (en) 2000-07-21 2005-10-28 Schering Corp Novel peptides as ns3-serine protease inhibitors of hepatitis c virus and pharmaceutical compositions containing them
US11021514B2 (en) 2016-06-01 2021-06-01 Athira Pharma, Inc. Compounds
GB2563396B (en) * 2017-06-12 2020-09-23 Ustav Organicke Chemie A Biochemie Av Cr V V I Inhibitors of Rhomboid Intramembrane Proteases
WO2022265577A2 (en) * 2021-06-15 2022-12-22 Agency For Science, Technology And Research Coronavirus enzyme modulators, methods of synthesis and uses thereof

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Abuelyaman, A.H., Flourescent derivatives of diphenyl [1-(N-Peptidylamino) alkyl] phosphonate Esters: synthesis and use in the inhibition and cellular localization of serine proteases, Bioconjugate Chemistry, vol. 5, No. 5, Oct. 1994 Washington, US, pp. 400-405, XP000465951.
Bonneau, P.B. et al, Evidence of conformational change in the human cytomegalovirus protease upon binding of peptidyl-activated carbonyl inhibitors, Biochemistry, vol. 36, No. 41, Oct. 14, 1997 Easton, PA, US, pp. 12640-12652.
Derstine, C.W., Trifluoromethyl-substituted imidazolines; novel precursors of trifluoromethyl ketones anenable to peptide synthesis, Journal of the American Chemical Society, vol. 228, No. 35, Sep. 4, 1996, pp. 8485-8486, XP 002065952.
Murphy, A..M. et al., Automated synthesis of Peptide C-Terminal aldehydes Journal of the American Chemical Society., vol. 114, No. 8, Apr. 8, 1992 pp. 3156-3157.
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Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6649593B1 (en) * 1999-10-13 2003-11-18 Tularik Inc. Modulators of SREBP processing
US6774212B2 (en) * 1999-12-03 2004-08-10 Bristol-Myers Squibb Pharma Company Alpha-ketoamide inhibitors of hepatitis C virus NS3 protease
US6599940B2 (en) 2000-09-13 2003-07-29 Georgetown University Synthesis of 2-hydroxymethylglutamic acid and congeners thereof
US20040092429A1 (en) * 2002-01-29 2004-05-13 Zealand Pharma A/S Compositions and methods for modulating connexin hemichannels
US7153822B2 (en) 2002-01-29 2006-12-26 Wyeth Compositions and methods for modulating connexin hemichannels
US20070042964A1 (en) * 2002-01-29 2007-02-22 Wyeth Compositions and methods for modulating connexin hemichannels

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